Cholera continues to be a major public health problem throughout the developing world. The primary objective of the original application was to test the hypothesis that environmental factors involving surface waters were responsible for the observed periodicity and pandemic nature of cholera. Data collected strongly suggest, but do not prove, that environmental factors are predictive of cholera outbreaks. Work will now focus on further defining the positive environmental associations that were found, and in determining, by genetic methodology, the relatedness of V. cholerae strains isolated from the environment and those from patients. Work will continue in Bangladesh in association with the Centre for Health and Population Research (ICDDR, B), the University of Maryland Biotechnology Institute and School of Medicine, and Emory University. Bi-weekly clinical and environmental surveillance will continue at two of the original sites where cholera outbreaks were frequent and in a new third site (Sunderbans), an area of mangrove swamps close to the Bay of Bengal, the site of entry for the O139 vibrios in Bangladesh in 1992 Improved assays will focus on: (1) identifying and culturing V. cholerae in surface waters, using both standard techniques and colonly blots with non-radioactive DNA probes, (for V. cholerae species, O1, O139, ctx, and tcpA) DNA extraction, and PCR, (2) assessing the relationship between clinical and environmental isolates by genotyping (using AFLP, ERIC-PCR, and MLST) and (3) identifying and enumerating V. cholerae attached to specific zooplankton species, using direct fluorescent antibody techniques and fluorescent in situ hybridization assays. Using the surveillance data our model of cholera transmission will be further refined. This will be useful in predicting outbreaks of cholera, not only in Bangladesh, but also in other countries with similar aquatic environments, thereby allowing early mobilization of preventive and treatment measures. Bangladesh is unique in affording the environment for doing these types of studies.